Dynamics of muscle fibre growth during postnatal mouse development

<p>Abstract</p> <p>Background</p> <p>Postnatal growth in mouse is rapid, with total skeletal muscle mass increasing several-fold in the first few weeks. Muscle growth can be achieved by either an increase in muscle fibre number or an increase in the size of individual myofibres, or a combination of both. Where myofibre hypertrophy during growth requires the addition of new myonuclei, these are supplied by muscle satellite cells, the resident stem cells of skeletal muscle.</p> <p>Results</p> <p>Here, we report on the dynamics of postnatal myofibre growth in the mouse extensor digitorum longus (EDL) muscle, which is essentially composed of fast type II fibres in adult. We found that there was no net gain in myofibre number in the EDL between P7 and P56 (adulthood). However, myofibre cross-sectional area increased by 7.6-fold, and length by 1.9-fold between these ages, resulting in an increase in total myofibre volume of 14.1-fold: showing the extent of myofibre hypertrophy during the postnatal period. To determine how the number of myonuclei changes during this period of intense muscle fibre hypertrophy, we used two complementary mouse models: <it>3F-nlacZ-E </it>mice express <it>nlacZ </it>only in myonuclei, while <it>Myf5</it>^{<it>nlacZ</it>/+}mice have β-galactosidase activity in satellite cells. There was a ~5-fold increase in myonuclear number per myofibre between P3 and P21. Thus myofibre hypertrophy is initially accompanied by a significant addition of myonuclei. Despite this, the estimated myonuclear domain still doubled between P7 and P21 to 9.2 × 10³μm³. There was no further addition of myonuclei from P21, but myofibre volume continued to increase, resulting in an estimated ~3-fold expansion of the myonuclear domain to 26.5 × 10³μm³by P56. We also used our two mouse models to determine the number of satellite cells per myofibre during postnatal growth. Satellite cell number in EDL was initially ~14 satellite cells per myofibre at P7, but then fell to reach the adult level of ~5 by P21.</p> <p>Conclusions</p> <p>Postnatal fast muscle fibre type growth is divided into distinct phases in mouse EDL: myofibre hypertrophy is initially supported by a rapid increase in the number of myonuclei, but nuclear addition stops around P21. Since the significant myofibre hypertrophy from P21 to adulthood occurs without the net addition of new myonuclei, a considerable expansion of the myonuclear domain results. Satellite cell numbers are initially stable, but then decrease to reach the adult level by P21. Thus the adult number of both myonuclei and satellite cells is already established by three weeks of postnatal growth in mouse.</p

Uncoordinated Transcription and Compromised Muscle Function in the Lmna-Null Mouse Model of Emery-Dreifuss Muscular Dystrophy

LMNA encodes both lamin A and C: major components of the nuclear lamina. Mutations in LMNA underlie a range of tissue-specific degenerative diseases, including those that affect skeletal muscle, such as autosomal-Emery-Dreifuss muscular dystrophy (A-EDMD) and limb girdle muscular dystrophy 1B. Here, we examine the morphology and transcriptional activity of myonuclei, the structure of the myotendinous junction and the muscle contraction dynamics in the lmna-null mouse model of A-EDMD. We found that there were fewer myonuclei in lmna-null mice, of which ∼50% had morphological abnormalities. Assaying transcriptional activity by examining acetylated histone H3 and PABPN1 levels indicated that there was a lack of coordinated transcription between myonuclei lacking lamin A/C. Myonuclei with abnormal morphology and transcriptional activity were distributed along the length of the myofibre, but accumulated at the myotendinous junction. Indeed, in addition to the presence of abnormal myonuclei, the structure of the myotendinous junction was perturbed, with disorganised sarcomeres and reduced interdigitation with the tendon, together with lipid and collagen deposition. Functionally, muscle contraction became severely affected within weeks of birth, with specific force generation dropping as low as ∼65% and ∼27% of control values in the extensor digitorum longus and soleus muscles respectively. These observations illustrate the importance of lamin A/C for correct myonuclear function, which likely acts synergistically with myotendinous junction disorganisation in the development of A-EDMD, and the consequential reduction in force generation and muscle wasting

Further Characterisation of the Molecular Signature of Quiescent and Activated Mouse Muscle Satellite Cells

Satellite cells are the resident stem cells of adult skeletal muscle. To date though, there is a paucity of native markers that can be used to easily identify quiescent satellite cells, with Pax7 probably being the best that is currently available. Here we have further characterized a number of recently described satellite cell markers, and also describe novel ones. Caveolin-1, integrin α7 and the calcitonin receptor proved reliable markers for quiescent satellite cells, being expressed by all satellite cells identified with Pax7. These three markers remained expressed as satellite cells were activated and underwent proliferation. The nuclear envelope proteins lamin A/C and emerin, mutations in which underlie Emery-Dreifuss muscular dystrophy, were also expressed in both quiescent and proliferating satellite cells. Conversely, Jagged-1, a Notch ligand, was not expressed in quiescent satellite cells but was induced upon activation. These findings further contribute to defining the molecular signature of muscle satellite cells

Integrated Functions of Pax3 and Pax7 in the Regulation of Proliferation, Cell Size and Myogenic Differentiation

Pax3 and Pax7 are paired-box transcription factors with roles in developmental and adult regenerative myogenesis. Pax3 and Pax7 are expressed by postnatal satellite cells or their progeny but are down regulated during myogenic differentiation. We now show that constitutive expression of Pax3 or Pax7 in either satellite cells or C2C12 myoblasts results in an increased proliferative rate and decreased cell size. Conversely, expression of dominant-negative constructs leads to slowing of cell division, a dramatic increase in cell size and altered morphology. Similarly to the effects of Pax7, retroviral expression of Pax3 increases levels of Myf5 mRNA and MyoD protein, but does not result in sustained inhibition of myogenic differentiation. However, expression of Pax3 or Pax7 dominant-negative constructs inhibits expression of Myf5, MyoD and myogenin, and prevents differentiation from proceeding. In fibroblasts, expression of Pax3 or Pax7, or dominant-negative inhibition of these factors, reproduce the effects on cell size, morphology and proliferation seen in myoblasts. Our results show that in muscle progenitor cells, Pax3 and Pax7 function to maintain expression of myogenic regulatory factors, and promote population expansion, but are also required for myogenic differentiation to proceed

Uncoordinated Transcription and Compromised Muscle Function in the Lmna-Null Mouse Model of Emery-Dreifuss Muscular Dystrophy

LMNA encodes both lamin A and C: major components of the nuclear lamina. Mutations in LMNA underlie a range of tissue-specific degenerative diseases, including those that affect skeletal muscle, such as autosomal-Emery-Dreifuss muscular dystrophy (A-EDMD) and limb girdle muscular dystrophy 1B. Here, we examine the morphology and transcriptional activity of myonuclei, the structure of the myotendinous junction and the muscle contraction dynamics in the lmna-null mouse model of A-EDMD. We found that there were fewer myonuclei in lmna-null mice, of which ∼50% had morphological abnormalities. Assaying transcriptional activity by examining acetylated histone H3 and PABPN1 levels indicated that there was a lack of coordinated transcription between myonuclei lacking lamin A/C. Myonuclei with abnormal morphology and transcriptional activity were distributed along the length of the myofibre, but accumulated at the myotendinous junction. Indeed, in addition to the presence of abnormal myonuclei, the structure of the myotendinous junction was perturbed, with disorganised sarcomeres and reduced interdigitation with the tendon, together with lipid and collagen deposition. Functionally, muscle contraction became severely affected within weeks of birth, with specific force generation dropping as low as ∼65% and ∼27% of control values in the extensor digitorum longus and soleus muscles respectively. These observations illustrate the importance of lamin A/C for correct myonuclear function, which likely acts synergistically with myotendinous junction disorganisation in the development of A-EDMD, and the consequential reduction in force generation and muscle wasting

Presenilin-1 acts via Id1 to regulate the function of muscle satellite cells in a γ-secretase-independent manner

Muscle satellite cells are the resident stem cells of adult skeletal muscle. Here, we have examined the role of the multifunctional protein presenilin-1 (PS1) in satellite cell function. PS1 acts as a crucial component of the γ-secretase complex, which is required to cleave single-pass transmembrane proteins such as Notch and amyloid-β precursor protein. PS1, however, also functions through γ-secretase-independent pathways. Activation of satellite cells was accompanied by induction of PS1, with PS1 knockdown enhancing their myogenic differentiation, but reducing their self-renewal. Transfection with siRNA against PS1 led to accelerated myogenic differentiation during muscle regeneration in vivo. Conversely, constitutive expression of PS1 resulted in the suppression of myogenic differentiation and promotion of the self-renewal phenotype. Importantly, we found that PS1 also acts independently of its role in γ-secretase activity in controlling myogenesis, which is mediated in part by Id1 (inhibitor of DNA binding 1), a negative regulator of the myogenic regulatory factor MyoD. PS1 can control Id1, which affects satellite cell fate by regulating the transcriptional activity of MyoD. Taken together, our observations show that PS1 is a key player in the choice of satellite cell fate, acting through both γ-secretase-dependent and γ-secretase-independent mechanisms

Emerin and lamin A/C are expressed in the nuclear envelope of quiescent satellite cells.

<p>Freshly isolated EDL myofibres were co-immunostained for Pax7 and emerin (a). Emerin (red) was expressed in the nuclear envelope of Pax7+ve (green) satellite cells (arrow). Since a mouse monoclonal antibody was used to immunostain lamin A/C, satellite cells were identified using caveolin-1 (b). We found that the nuclear lamina of caveolin-1 (red)-expressing satellite cells (arrow) contained lamin A/C (green). All nuclei present were revealed by counterstaining with DAPI (blue). Scale bar represents 20 µm.</p